WO2015127365A2 - Mutants de sortase a calcium-indépendants - Google Patents

Mutants de sortase a calcium-indépendants Download PDF

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Publication number
WO2015127365A2
WO2015127365A2 PCT/US2015/017116 US2015017116W WO2015127365A2 WO 2015127365 A2 WO2015127365 A2 WO 2015127365A2 US 2015017116 W US2015017116 W US 2015017116W WO 2015127365 A2 WO2015127365 A2 WO 2015127365A2
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seq
mutant
amino acid
sortase
calcium
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PCT/US2015/017116
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English (en)
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WO2015127365A3 (fr
Inventor
Jessica Ingram
Hidde Ploegh
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Whitehead Institute For Biomedical Research
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/2207—Sortase A (3.4.22.70)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • Protein engineering is becoming a widely used tool in many areas of protein biochemistry.
  • One engineering method is controlled protein ligation.
  • Native chemical protein ligation relies on efficient preparation of synthetic peptide esters, which can be technically difficult to prepare for many proteins.
  • Recombinant technologies can be used to generate protein-protein fusions, joining the C-terminus of one protein with the N-terrninus of another protein.
  • Intein-based protein ligation systems can also be used to join proteins.
  • a prerequisite for this intein-mediated ligation method is that the target protein is expressed as a correctly folded fusion with the intein, which is often
  • the transpeptidation reaction catalyzed by sortases has emerged as a general method for derivatizing proteins with various types of modifications.
  • target proteins are engineered to contain a sortase recognition motif (LPXT) near their C- termini .
  • LXT sortase recognition motif
  • these artificial sortase substrates undergo a transacylation reaction resulting in the exchange of residues C-terminal to the threonine residue with the synthetic oligoglycine peptide , resulting in the protein C-terminus being ligated to the N- terminus of the synthetic peptide .
  • SaSrtA S . aureus sortase A
  • Ca 2+ dependency that can limit the effectiveness of SaSrtA in low Ca 2+ concentrations , such as the cytoplasm or when Ca 2+ binding compounds, for example phosphate, carbonate, and
  • EDTA ethylenediaminetetraacetic acid
  • the disclosure provides a sortase A mutant comprising at least three amino acid substitutions relative to a wild- type sortase A, wherein the amino acid substitutions comprise a) a K residue at position 105 ; b) a Q or A residue at position 108 ; and c) at least one amino acid substitution selected from the group
  • the wild-type sortase A comprises a S. aureus sortase A
  • the wild-type sortase A comprises a protein sequence of SEQ ID NO: 1.
  • the wild-type sortase A comprises a protein sequence of SEQ ID NO: 3.
  • the wild- type sortase A comprises a protein of SEQ ID NO: 5.
  • the mutant comprises a deletion of amino acids 2-25. In some embodiments, comprising a deletion of amino acids 2-59.
  • the mutant comprises at least two amino acid substitutions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least three amino acid substitutions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least four amino acid substitu ions selected from the group consisting of i) -vi) . In some embodiments, the mutant comprises at least five amino acid substitutions selected from the group consisting of i) -vi) .
  • the mutant comprises at least 60% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises at least 80% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises at least 90% sequence identity to amino acid residues 60-206 of the wild-type sortase A. In some embodiments, the mutant comprises one or more C- terminal or N-terminal tags. In some embodiments, the one or more C-terminal or N-terminal tags comprises a His6 tag.
  • the mutant exhibits sortase A catalytic activity in the absence of calcium.
  • the mutant exhibits sortase A catalytic activity in the absence of exogenous calcium. In some embodiments, the mutant exhibits sortase A catalytic activity in the presence of calcium-binding proteins. In some embodiments, the mutant exhibits sortase A catalytic activity in the presence of calcium concentrations up to 10 m .
  • the disclosure provides a
  • the disclosure provides a polynucleotide encoding a catalytically active variant or fragment of a mutant srtA described herein.
  • the disclosure provides a nucleic acid construct comprising the polynucleotides described herein .
  • the disclosure provides a host cell transformed with the nucleic acid constructs described herein .
  • the disclosure provides a method of preparing a mutant sortase A comprising: (a) culturing the host cell of claim 22 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally (b) purifying the mutant sortase A to provide a mutant sortase A.
  • the disclosure provides an enzyme composition comprising at least one sortase A mutant described herein. In some aspects, the disclosure provides a method comprising performing a sortase-mediated transpeptidation reaction catalyzed by the enzyme compositions described herein .
  • the disclosure relates to the use of an enzyme composition described herein for the sortagging of a target protein.
  • the disclosure provides a method for sortagging a target protein, comprising: (a)
  • transamidate the target protein and the moiety thereby sortagging the target protein.
  • the terminal oligoglycine sequence comprises 1-10 N-terminal glycine residues.
  • the moiety comprises an amino acid, a peptide, a protein, a polynucleotide, a
  • a tag a metal atom, a chelating agent, a contrast agent, a catalyst, a polymer, a recognition element, a small molecule, a lipid, a label, an epitope, a small molecule, a therapeutic agent, a cross- linker, a toxin, a radioisotope, an antigen, or a click chemistry handle .
  • the disclosure provides a kit for sortagging a target protein comprising the enzyme composition of claim 24.
  • the disclosure provides a sortase A mutant comprising an amino acid sequence at least 80% identical to SEQ ID NO : 9, and wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO : 9 ; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO: 9; and v) a T residue at position 138 of SEQ ID NO: 9.
  • the mutant comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 21.
  • the mutant comprises at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 15.
  • the mutant comprises at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, the mutant comprises an amino acid sequence of SEQ ID NO: 14.
  • the mutant comprises at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments , the mutant comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, the mutant comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 96% identical to SEQ ID NO: 9. In some
  • the mutant comprises an amino acid sequence at least 97% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 97% identical to SEQ ID NO: 9.
  • the mutant comprises an amino acid sequence at least 98% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 98% identical to SEQ ID NO: 9.
  • the mutant comprises an amino acid sequence at least 99% identical to SEQ ID NO: 9. In some embodiments, the mutant comprises an amino acid sequence at least 99% identical to SEQ ID NO: 9.
  • the mutant comprises an amino acid sequence of SEQ ID NO: 9.
  • the disclosure comprises an enzyme composition comprising at least one mutant sortase A described herein.
  • the at least one mutant sortase A is selected from the group consisting of SEQ ID NOs : 9-21.
  • the at least one mutant sortase A is selected from the group consisting of catalytically active variants of SEQ ID NOs : 9-21.
  • the at least one mutant sortase A is selected from the group consisting of catalytically active fragments of SEQ ID NOs : 9-21.
  • the disclosure provides a method comprising performing a sortase-mediated transpeptidation reaction catalyzed an enzyme composition described herein .
  • the disclosure relates to the use of an enzyme composition described herein for the sortagging of a target protein.
  • the disclosure relates to a method for sortagging a target protein, comprising: (a) providing a target protein comprising a sortase
  • the oligoglycine sequence comprises 1-10 N-terminal glycine residues.
  • the moiety comprises an amino acid, a peptide, a protein, a polynucleotide, a
  • a tag a metal atom, a chelating agent, a contrast agent, a catalyst, a polymer, a recognition element, a small molecule, a lipid, a label, an epitope, a small molecule, a therapeutic agent, a cross- linker, a toxin, a radioisotope, an antigen, or a click chemistry handle .
  • the disclosure provides a kit for sortagging a target protein comprising an enzyme composition described herein.
  • the disclosure provides a
  • polynucleotide encoding a sortase A mutant comprising a nucleotide sequence at least 80% identical to SEQ ID NO. 10, wherein the nucleotide sequence encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a Q or A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138.
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 selected from the group
  • polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) - v) .
  • the polynucleotide comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 10.
  • the polynucleotide comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, the polynucleotide comprises a nucleotide sequence of SEQ ID NO : 10.
  • the disclosure provides a nucleic acid construct comprising any of the polynucleotides described herein.
  • the nucleic acid construct comprises a nucleotide sequence that encodes one or more C-terminal or N-terminal tags.
  • the nucleic acid construct comprises one or more C-terminal or N-terminal tags comprises a His6 tag .
  • the disclosure provides a host cell transformed with a nucleic acid construct described herein .
  • the disclosure provides a method of preparing a mutant sortase A comprising: (a) culturing the host cell of claim 75 in a suitable culture medium under suitable conditions to produce the mutant sortase A; and optionally (b) purifying the mutant sortase A to provide a mutant sortase A.
  • FIG. 1 is a schematic representation of a sortase- catalyzed transacylation reaction.
  • FIG . 2 is a schematic representation of the site- specific C-terminal labeling scheme (left) and N-terminal labeling scheme (right) using sortase A.
  • labeling begins with a substrate- recognition step (top) , and then proceeds with generation of a thioacyl intermediate (middle) , followed by ligation of an exogenously
  • FIGS . 3A-3H are schematic representations of various polypeptide conjugations using sortase-mediated ligation .
  • FIG . 3A is a schematic representation showing the sortagging of a molecular probe carrying an oligoglycine tag to a target pro ein having a C- erminal LPXTG- tag .
  • FIG. 3B is a schematic representation showing sortase-mediated ligation of a polypeptide to a nucleic acid .
  • FIG. 3C is a schematic representation of a sortase- mediated ligation being used to produce a neoglyconjugate by fusing a polypeptide comprising the LPXTG sortase recognition motif to amino-methylene groups in 6- aminohexoses .
  • FIG. 3D is a schematic representation of the immobilization of an LPXTG- tagged protein onto an oligoglycine-coated solid surface, for example,
  • FIG. 3E is a schematic representation of a cell surface protein genetically engineered to include an extracellular C-terminal region expressing a C-terminal LPXTG motif, which has been labeled with a triglycine- tagged probe by sortagging.
  • FIG. 3F is a schematic illustration of the
  • dimerization/oligomerization of a protein by sortagging of a bifunctional protein possessing an N-terminal oligoglycine tag and a C-terminal LPXTG tag.
  • FIG . 3G is a schematic representation of the circularization of a bifunctional protein containing an N-terminal oligoglycine tag and a C-terminal LPXTG tag utilizing sortase-mediated ligation.
  • FIG . 3H is a schematic representation of the site- specific attachment of a lipid utilizing sortase-mediated transpeptidation .
  • FIGS . 4A-4U show exemplary sequences contemplated by the present disclosure . Mutations relative to wild- type sortase A are shown in red font . Portions of sequences that are absent in an exemplary wild-type S. aureus sortase A sequence and/or contain a 6XHis tag are highlighted in yellow .
  • FIG. 4A is an exemplary wild type S. aureus Sortase A (SaSrtA) amino acid sequence.
  • FIG. 4B is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4A.
  • FIG. 4C is an exemplary wild type S. aureus Sortase A (SaSrtA) amino acid sequence.
  • FIG. 4D is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4C.
  • FIG. 4E is an exemplary wild type sortase delta 25 amino acid sequence.
  • FIG. 4F is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4E.
  • FIG. 4G is an exemplary mutant srtA
  • FIG. 4H is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG. 4G.
  • FIG. 41 is an exemplary calcium- independent mutant srtA P94R/D160N/D165A/K190E/K196T amino acid sequence.
  • FIG. 4J is an exemplary nucleotide sequence encoding the amino acid sequence shown in FIG . 41.
  • FIG . 4K is an exem lary calcium- independent srtA P94S/D160N/D165A/K196T amino acid sequence.
  • FIG. 4L is an exemplary calcium-independent srtA
  • FIG. 4M is an exemplary calcium- independent srtA P94S/D160N/K196T amino acid sequence.
  • FIG. 4N is an exemplary calcium- independent srtA P94S/D160N/D165A amino acid sequence.
  • FIG . 40 is an. exemplary calcium-independent srtA P94S/D165A amino acid sequence .
  • FIG. 4P is an exemplary calcium-independent srtA P94S amino acid sequence.
  • FIG. 4Q is an exemplary calcium-independent srtA P94R amino acid sequence.
  • FIG. 4R is an exemplary calcium- independent srtA
  • FIG. 4S is an exemplary calcium- independent srtA D165A amino acid sequence.
  • FIG. 4T is an exemplary calcium- independent srtA K190E amino acid sequence.
  • FIG. 4U is an exemplary calcium- independent srtA K196T amino acid sequence.
  • FIGS. 5A and 5B show exemplary alignments of various srtA sequences contemplated by the present disclosure.
  • FIG. 5A shows an alignment of SEQ ID NO: 1 and SEQ
  • SEQ ID NO: 9 indicating that SEQ ID NO: 9 is at least 65% identical to SEQ ID NO : 1.
  • FIG. 5B shows an alignment of SEQ ID NO: 7 and SEQ ID NO: 9, indicating that SEQ ID NO: 9 is at least 98% identical to SEQ ID NO: 7.
  • FIG. 6 shows a comparison of activities of various sortases at 0 °C, in the presence and absence of calcium.
  • 30uM of a substrate (VHH84D4) containing an LPETGG- 6xHis C-terminal tag was incubated in 50mM Tris, pH 7.5, 150mM NaCl , 500uM GGG-TAMRA and lOmM CaC12 or lOmM EGTA with either 5uM (1) WT SrtA delta 25, (2) 5uM pentamutant SrtA or (3) heptamutant SrtA for up to 6 hours at 0°C.
  • the reaction was then visualized by running a sample on a 12% Tris-glycine SDS PAGE gel.
  • FIGS. 7A - 7C are chromatograms demonstrating that the calcium-independent srtA mutant of SEQ ID NO: 9 exists predominantly in monomeric form (FIG. 7A) compared to the calcium-dependent srtA mutant of SEQ ID NO: 7 which exists in both dimeric and monomeric form (FIG. 7B) , and the calcium-dependent wild-type srtA which also exists in both dimeric and monomeric form (FIG. 7C) .
  • FIG. 8 is a photograph of a gel demonstrating the high purity of monomeric sortase.
  • FIG. 9 is a chromatogram demonstrating the stability of monomeric sortase. DETAILED DESCRIPTION OF THE INVENTION
  • aspects of the disclosure relate to sortase mutants, and in particular to sortase A mutants that exhibit an indifference to calcium. Work described herein
  • sortase A mutants described herein surprisingly and unexpectedly exhibit sortase A catalytic activity (e.g., increased catalytic activity compared to wild type sortase A, ) , and do so in a manner that is independent of the presence or concentration of calcium.
  • Sortases, sortase-mediated transacylation reactions, and their use in transacylation (sometimes also referred to as transpeptidation) for protein engineering are well known to those of skill in the art (see, e.g., Ploegh et al., International Patent Application PCT/US2010/000274 , and Ploegh et al . , International Patent Application
  • FIG. 1 shows an exemplary transpeptidation reaction catalyzed by sortase, which results in the ligation of species containing a sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LPXT motif or an LPXTG motif) with those bearing an N-terminal sortase recognition motif (e.g., an LP
  • sortases can be used for both C- terminal (left) and N-terminal (right) site-specific labeling.
  • the sortase transacylation reaction allows for the facile installation of all kinds of substituents at the C- terminus or N-terminus of a suitably modified protein.
  • the sortase reaction can be employed for ligating polypeptides to one another, ligating synthetic peptides to recombinant proteins, linking a reporting molecule to a polypeptide, linking a polypeptide to a label or probe (FIG. 3A) , joining a nucleic acid to a polypeptide (FIG. 3B) , ligating a glycan to a polypeptide (FIG. 3C) , conjugating a polypeptide to a solid support or polymer (FIG. 3D) , site- specific modification of the extracellular C-terminal region of cell surface proteins expressed in living cells (FIG. 3E) , site- specific modification of the extracellular N-terminal region of cell surface proteins expressed in living cells ,
  • sortase A derives its ability to site- specifically modify target proteins by recognition of sortase recognition motifs, such as the motif LPXTG.
  • sortase recognition motifs such as the motif LPXTG.
  • Other suitable sortase recognition motifs are apparent to the skilled artisan. It will be
  • a recognition sequence with respect to sequences recognized by sortase, are used interchangeably.
  • a recognition sequence further comprises one or more additional amino acids, e.g., at the N or C terminus. Such additional amino acids may provide context that improves the recognition of the recognition motif.
  • sortase recognition sequence may refer to a masked or unmasked sortase recognition sequence.
  • a calcium-independent mutant sortase has a different substrate specificity with regard to the sortase recognition motif as compared to a calcium-dependent wild type sortase.
  • Sortases with different substrate specificities with regard to the sortase recognition motif recognize different sortase recognition sequences. For example, Sortases 1 and 2 are said to have different substrate specificities if SRM1 and SRM2 are different sortase recognition motifs and Sortase 1 recognizes SRM1 and Sortase 2 is active in recognizing SRM2 but has little or no activity
  • the substrate specificities overlap in that one of the two sortases recognizes both SRM1 and SR 2 while the other sortase recognizes only one of the SRMs . In some embodiments the substrate specificities do not overlap, e.g., Sortase 1 recognizes SRM1 but does not recognize SRM2 , and Sortase 2 recognizes SRM2 but does not recognize SRM1. In some embodiments, two calcium- independent sortases may alternately or additionally utilize different
  • nucleophiles are nucleophiles . In some embodiments the nucleophile specificity overlaps while in some embodiments the nucleophile specificity does not overlap.
  • a sortase with an altered substrate specificity with regard to the sortase recognition motif may be generated by engineering one or more mutations in the sortase, e.g., in a region of the protein that is involved in recognition and/or binding of the sortase recognition motif, e.g., the putative substrate recognition loop (e.g., the loop connecting strands ⁇ 6 and ⁇ 7 ( ⁇ 6/ ⁇ 7 loop) in SrtA (Val 161 -Asp 17S ) .
  • the putative substrate recognition loop e.g., the loop connecting strands ⁇ 6 and ⁇ 7 ( ⁇ 6/ ⁇ 7 loop) in SrtA (Val 161 -Asp 17S ) .
  • a phage- display, yeast display, or other screen of a mutant sortase library randomized in the substrate recognition loop may be performed, and variants with altered
  • a sortase with an altered nucleophile specificity may be generated by engineering one or more mutations in the sortase, e.g., in a region of the protein that is involved in
  • a phage-display screen of a mutant sortase library randomized in the substrate recognition loop may be performed, and variants with altered substrate specificity may be identified.
  • a calcium- independent sortase described herein is modified to alter its substrate specificity with regard to the sortase recognition motif and/or its nucleophile specificity by introducing one or more mutations into the sortase.
  • a calcium-dependent sortase is modified to alter its substrate specificity and/or nucleophile specificity and is rendered calcium- independent by introducing the mutations described herein. It will be appreciated that mutations conferring calcium- independence and altered substrate and/or nucleophile specificity may be engineered individually or sequentially in groups of one or more, in any order or combination that results in a desired sequence. In some embodiments two calcium- independent sortases with different substrate
  • nucleophile specificities and/or different nucleophile specificities are derived from the same wild type sortase, e.g., S. aureus SrtA.
  • the substrate e.g., S. aureus SrtA.
  • Sortases with different substrate specificities may be used, for example, to introduce two different moieties to a target protein.
  • a target protein may comprise or be modified to comprise first and second SRMs.
  • Two sortases each of which specifically recognizes only one of the two SRMs, may be used to conjugate two agents to the protein by reaction with the two SRMs, e.g., at the N- and C- termini .
  • the conjugations may take place in a single reaction vessel or may be performed sequentially.
  • the two agents may be any of the agents described herein, and may be the same or different.
  • a first agent may comprise a toxin and a second agent may comprise a detectable label , e.g., for imaging.
  • the disclosure provides a kit comprising two calcium- independent sortases with
  • nucleophile specificity is derived from the same wild type sortase , e.g., S . aureus SrtA.
  • substrate specificities e.g., S . aureus SrtA.
  • substrate specificities e.g., S . aureus SrtA.
  • amino acid sequences of sortase A polypeptides and the nucleotide sequences that encode them are known to those of skill in the art and are disclosed in a number of references cited herein, the entire contents of all of which are incorporated herein by reference.
  • polypeptide refers to a molecule comprising at least two covalently attached amino acids.
  • a polypeptide can be made up of naturally occurring amino acids and peptide bonds and/or synthetic peptidomimetic residues and/or bonds.
  • Polypeptides described herein include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial (e.g., E. coli) , yeast, higher plant, insect and mammalian cells.
  • the disclosure also features biologically active fragments of calcium- independent srtA mutants.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline .
  • Amino acid analogs are compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, e.g., norleucine. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid .
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.
  • Sortase A is a polypeptide having a length of 206 amino acids which typically comprises a hydrophobic N- terminal domain (e.g., residues 1 to about 25) which functions as both a signal peptide and a membrane anchoring domain, a central linker domain (e.g., from about residue 26 to about residue 59) , and a C-terminal catalytic domain (e.g., from about residue 60 to about residue 206) .
  • a hydrophobic N- terminal domain e.g., residues 1 to about 25
  • a central linker domain e.g., from about residue 26 to about residue 59
  • C-terminal catalytic domain e.g., from about residue 60 to about residue 206 .
  • FIG. 4A shows an exemplary nucleotide sequence (SEQ ID NO: 2; NCBI
  • NC_002745.2 encoding the wild type SaSrtA protein.
  • FIG. 4C Another exemplary sequence of wild- type S. aureus SrtA is shown in FIG. 4C (SEQ ID NO: 3; GenBank Accession number AAD48437 ; NCBI Reference Sequence:
  • wild-type S. aureus SrtA sequences, calcium- independent srtA mutants, and catalytically active fragments or variants thereof disclosed herein may comprise either a K or N at position 57. It should be appreciated that wild-type S. aureus SrtA sequences, calcium- independent srtA mutants, and catalytically active fragments or variants thereof disclosed herein may comprise either an E or G at position 167.
  • aspects of the present disclosure relate to calcium- independent sortase A mutants. Such mutants may be produced through processes such as directed evolution, site-specific modification, etc . It should be
  • the calcium- independent srtA mutants disclosed herein can be used in any application in which sortagging is desirable, including for example, the sortase -mediated ligation reactions described in FIGS. 3A-3I .
  • the calcium- independent srtA mutants comprise at least one amino acid substitution relative to a wild-type sortase A polypeptide, and catalytically active fragments, catalytically active derivatives, or catalytically active variants thereof, and polynucleotides encoding the same.
  • calcium- independent refers to the ability of a sortase A enzyme to exhibit catalytic activity in a manner that is independent of the presence of calcium, or independent of the amount of calcium present within a concentration range that is not
  • the calcium- independent srtA mutants disclosed herein can be assayed for their ability to exhibit sortase A catalytic activity in a calcium- independent manner by contacting a target protein comprising a C-terminal sortase
  • the calcium- independent srtA mutants can be assayed for their ability to exhibit sortase A catalytic activity in the presence of calcium
  • calcium-dependent sortase A concentrations which are required for calcium-dependent sortase A to exhibit catalytic activity.
  • "calcium-dependent" in connection with a sortase means that the catalytic activity of the sortase relies or depends on the presence and concentration of calcium, such that in the absence of calcium or the absence of a sufficient amount of calcium, the calcium-dependent sortase will not exhibit sortase A catalytic activity or has greatly reduced catalytic activity as compared with its activity when calcium is present in sufficient amounts (e.g., 5 mM - 10 mM) .
  • the present disclosure contemplates any srtA variants which exhibit sortase A catalytic activity comparable to the wild-type srtA enzyme, as long as they possess calcium- independent sortase A catalytic activity.
  • fragments As used herein in connection with “fragments” ,
  • sortase A catalytic activity refers to the ability of a sortase to catalyze the cleavage of a polypeptide within a sortase A consensus recognition sequence and ligate the free primary amino group (NH 2 -CH 2 -) of a oligoglycine sequence to the free C-terminal carboxyl group of the cleaved polypeptide.
  • oligoglycine refers to a (Gly) n s equence, wherein n is between 1 and about 10, or more preferably between 2 and about 5, and even more preferably 2 or 3 , glycine residues.
  • An "N-terminal" oligoglycine sequence is located at the N-terminus of a polypeptide , such that the polypeptide comprises a free primary amino (NH 2 personallyCH 2 _) group at its N- erminus .
  • N- terminal oligoglycine sequence can also include an internal oligoglycine sequence that is capable of forming a oligoglycine sequence under applicable conditions , e.g., by cleavage of an N-terminal peptide sequence by an endogenous host cell enzyme, or by specific proteolytic cleavage in vitro .
  • Sortase A catalytic activity of the calcium- independent srtA mutants disclosed herein can be assayed using methods known in the art. The crystal structure of SrtA complexed with a substrate has been determined allowing catalytic active domains of sortase A proteins from various Gram-positive bacterium to be easily discerned by those of skill in the art, see for example, Y. Zong et al . J. Biol Chem. 2004, 279, 31383-31389, which is incorporated herein by reference.
  • polynucleotide has one or more alterations (e.g., additions, substitutions, and/or deletions) with respect to a reference polypeptide or polynucleotide, which may be referred to as the "original polypeptide” or “original polynucleotide", respectively.
  • An addition may be an insertion or may be at either terminus.
  • a variant may be shorter or longer than the reference polypeptide or polynucleotide.
  • variant encompasses
  • fragments are continuous portions of a polypeptide or polynucleotide that is shorter than the reference polypeptide or polynucleotide.
  • a variant comprises or consists of a
  • a fragment or variant is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or more as long as the reference polypeptide or polynucleotide.
  • a fragment may lack an N-terminal and/or C-terminal portion of a reference polypeptide.
  • a fragment may lack up to 5%, 10%, 15%, 20%, or 25% of the length of the polypeptide from either or both ends.
  • a fragment may be an N-terminal, C-terminal, or internal fragment.
  • a variant polypeptide comprises or consists of at least one domain of a reference polypeptide.
  • a variant polynucleotide hybridizes to a reference polynucleotide under art-recognized stringent conditions, e.g., high stringency conditions, for sequences of the length of the reference polypeptide.
  • a variant polypeptide or polynucleotide comprises or consists of a polypeptide or polynucleotide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the reference polypeptide or polynucleotide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide or polynucleotide.
  • a variant polypeptide comprises or consists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical in sequence to the reference polypeptide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide, with the proviso that, for purposes of computing percent identity, a conservative amino acid substitution is considered identical to the amino acid it replaces .
  • a variant polypeptide comprises or consists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more identical to the reference polypeptide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the reference polypeptide, with the proviso that any one or more amino acid substitutions (up to the total number of such substitutions) may be restricted to conservative substitutions.
  • a percent identity is measured over at least 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000;
  • sequence of a variant polypeptide comprises or consists of a sequence that has N amino acid differences with respect to a reference sequence, wherein N is any integer between 1 and 10 or between 1 and 20 or any integer up to 1%, 2%, 5%, or 10% of the number of amino acids in the reference polypeptide, where an "amino acid difference" refers to a substitution, insertion, or deletion of an amino acid.
  • a difference is a conservative substitution. Conservative substitutions may be made, e.g., on the basis of
  • a variant is a functional variant, i.e., the variant at least in part retains at least one activity (e.g., calcium- independent sortase A catalytic activity) of the reference polypeptide or polynucleotide. In some embodiments a variant at least in part retains more than one or substantially all known activities of the reference polypeptide or
  • an activity may be, e.g., a catalytic activity, binding activity, ability to perform or participate in a biological function or process, etc.
  • an activity is one that has (or the lack of which has) a detectable effect on an observable phenotype of a cell or organism.
  • an activity of a variant may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more, of the activity of the reference polypeptide or polynucleotide , up to approximately 100%, approximately 125%, or approximately 150% of the activity of the reference polypeptide or polynucleotide, in various embodiments .
  • a variant e.g., a functional variant, comprises or consists of a polypeptide at least 80%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%. 99.5% or 100% identical to an reference polypeptide or polynucleotide over at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or 100% of the full length of the reference polypeptide or polynucleotide or over at least 70%, 75%, 80%, 85%, 90%, 92.5%, 95%, 96%, 97%, 98%, or 99% or 100% of a functional fragment of the reference polypeptide or polynucleotide.
  • an alteration e.g., a substitution or deletion, e.g., in a functional variant, does not alter or delete an amino acid or nucleotide that is known or predicted to be important for an activity, e.g., a known or predicted catalytic residue or residue involved in binding a substrate or cofactor.
  • nucleotide (s) amino acid(s), or region (s) exhibiting lower degrees of conservation across species as compared with other amino acids or regions may be selected for alteration.
  • Variants may be tested in one or more suitable assays to assess activity.
  • a polypeptide or polynucleotide sequence in the NCBI RefSeq database may be used as a reference sequence.
  • polynucleotide is a naturally occurring variant or fragment. In some embodiments a variant or fragment of a naturally occurring polypeptide or polynucleotide is not naturally occurring.
  • the calcium-independent srtA mutant is selected according to the degree of sequence homology with a wild-type sortase A enzyme. In some embodiments , the calcium- independent srtA mutant is selected according to the degree of sequence homology with S. aureus sortase A. In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 1 (FIG. 4A) . In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 3 (FIG. 4C) . In some
  • the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 5 (FIG. 4E) . In some embodiments, the calcium- independent srtA mutant is selected according to the degree of sequence homology with SEQ ID NO: 7 (FIG. 4G) .
  • Calcium- independent srtA mutants having a desired degree of homology to a wild-type sortase A enzyme can be identified by, e.g., using the wild-type sortase A nucleotide sequences as query sequences in a search against public databases to identify related sequences.
  • the calcium- independent srtA mutant comprises an amino acid sequence homologous to amino acids 60-206 of SEQ ID NO: 1, e.g. an amino acid sequence that is at least 55%, at least 60%, at least
  • the calcium- independent srtA mutant comprises an amino acid sequence homologous to amino acids 60-206 of SEQ ID NO: 3, e.g. an amino acid sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or higher, homologous thereto.
  • a nucleotide sequence encoding a calcium- independent srtA mutant has at least 25%, or preferably at least 30%, or more preferably at least 35% or more identity with the nucleic acid sequence of SEQ ID NO : 2.
  • a nucleotide sequence encoding a calcium- independent srtA mutant has at least 25%, or preferably at least 30%, or more preferably at least 35% or more identity with the nucleic acid sequence of SEQ ID NO: 4.
  • the calcium- independent srtA mutant has at least 35%, or preferably at least 40%, or more preferably at least 45% similarity with the amino acid sequence of SEQ ID NO: 1. In further embodiments, the calcium- independent srtA mutant has at least 35%, or preferably at least 40%, or more preferably at least 45% similarity with the amino acid sequence of SEQ ID NO: 3.
  • the calcium-independent srtA mutant is a variant of SrtA of another Gram-positive bacterium having one or more as substitutions, deletions, insertions , and/or other modifications relative to the native nucleotide and/or amino acid sequence of S . aureus srtA.
  • the variant comprises one or more conservative amino acid substitutions relative to SrtA of another Gram-positive bacterium.
  • the variant comprises one or more amino acid substitutions relative to SrtA of another Gram-positive bacterium, wherein the one or more as amino acid
  • substitutions are predominantly, e.g., at least 50% , or preferably at least 60%, or more preferably at least 70% or more , conservative substitutions .
  • fragments of calcium- independent srtA mutants disclosed herein exhibiting calcium- independent sortase A catalytic activity are contemplated herein, and can be utilized in the methods described herein.
  • fragments can be identified by producing transaminase fragments by known recombinant techniques or proteolytic techniques , for example , and determining the rate of protein or peptide ligation.
  • the fragment sometimes consists of about 80% of the full-length transamidase amino acid sequence, and sometimes about 70%, about 60%, about 50%, about 40% or about 30% of the full-length transamidase amino acid sequence such as that of a wild- type S. aureus Sortase A.
  • the fragment lacks an N-terminal portion of the full-length sequence, e.g., the fragment lacks the N-terminal portion extending to the end of the membrane anchor sequence.
  • the fragment comprises the C-terminus of a full-length transamidase amino acid sequence.
  • a catalytic core region from a sortase is utilized, e.g., a region from about position 60 to about position 206 of SrtA, e.g., S. aureus SrtA.
  • the fragment comprises a sortase A lacking N-terminal amino acids 2-25 (SEQ ID NO: 5) (FIG. 4E) .
  • An exemplary nucleotide sequence i.e., SEQ ID NO: 6) encoding such fragment is shown in FIG. 4F.
  • Calcium-independent srtA mutants can be derived from sortase A sequences from other organisms.
  • the disclosure provides mutants of any Ca ++ - dependent SrtA from a species other than S. aureus, wherein the mutants comprise any of the mutations or combinations of mutations described herein at the corresponding positions in the Ca' " -dependent SrtA from a species other than S. aureus.
  • a calcium- independent srtA mutant is derived from sortase A sequences from other organisms comprising nucleotide sequences substantially identical or similar to the nucleotide sequences that encode Srt A.
  • a similar or substantially identical nucleotide sequence may include modifications to the native sequence, such as
  • nucleotide sequences that sometimes are about at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more identical to a native nucleotide sequence, and sometimes are about 90% or 95% or more identical to the native nucleotide sequence (each identity percentage can include a 1%, 2%, 3% or 4% variance) .
  • One test for determining whether two nucleic acids are substantially identical is to determine the percentage of identical nucleotide sequences shared between the nucleic acids.
  • sequence identity can be performed by a variety of techniques which are available to the skilled artisan. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., may be used to generate alignments and/or to obtain a percent identity.
  • computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, etc., may be used to generate alignments and/or to obtain a percent identity.
  • a calcium- independent srtA mutant comprises at least three amino acid substitutions relative to a wild-type sortase A, wherein the amino acid substitutions comprise a) a K residue at position 105; b) a Q or A residue at position 108; and c) at least one amino acid substitution selected from the group
  • mutant is used to refer to a one amino acid sequence which has changed by at least one amino acid residue relative to another amino acid sequence.
  • the term "mutant” with reference to “sortases” should not be considered to imply that any particular way of generating the mutant sequences is required or that any particular starting materials is required .
  • the present disclosure contemplates any suitable method of generating the calcium-independent srtA mutants described herein.
  • suitable methods include, but are not limited to introducing mutations into an appropriate wild-type coding sequence, synthesizing the sequences of the calcium-independent srtA mutants de novo, for example, utilizing solid phase peptide synthesis, and in vitro translation a synthetic mRNA, to name only a few. It is to be further understood that the disclosure contemplates calcium- independent mutants of any wild-type sortase A. Those skilled in the art will appreciate that the wild- type sequences of sortase A may vary, e.g., SrtA from various species may have gaps, insertions, and/or vary in length relative to the amino acid sequence of exemplary wild-type S. aureus SrtA.
  • the disclosure is not intended to be limited in any way by the original amino acid residue at a particular position in any wild- type sortase A sequence used to generate a calcium- independent srtA mutant.
  • any substitution which results in the specified amino acid residue at a position specified herein is contemplated by the disclosure.
  • the phrase "X residue at position Y" means that the X residue in the resulting mutant srtA replaces whatever amino acid was present in the original sortase A amino sequence at the position Y in the original sorta.se A amino acid sequence that corresponds to the same position in an exemplary wild-type S.
  • aureus srtA amino acid sequence when accounting for any gaps and/or insertions in the original sortase A amino acid sequence relative to the exemplary wild-type S. aureus srtA amino acid sequence.
  • the following examples are instructive and are not intended to be limiting in any way . If the original wild-type sortase A used to generate a srtA mutant is the wild-type S. pyogenes srtA (NCBI Gene ID: 901269), "position 94" in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 115 of the wild-type S. pyogenes srtA as position 115 of the wild-type S .
  • pyogenes srtA corresponds to position 94 in the exemplary wild-type S. aureus srtA sequence (NCBI Gene ID: 3238307) when taking into account gaps and/or insertions in the sequence alignment of S. pyogenes srtA and S. aureus srtA.
  • N residue at position 115 of the wild- type S. pyogenes srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.
  • position 94 in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 123 of the wild-type B .
  • anthracis srtA as position 123 of the wild-type B .
  • anthracis srtA corresponds to position 94 in the exemplary wild-type S. aureus srtA sequence (NCBI Gene ID: 3238307) when taking into account gaps and/or insertions .in the sequence alignment of B . anthracis srtA and S . aureus srtA.
  • the E residue at position 123 of the wild- type B . anthracis srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.
  • position 94 in the phrase "an amino acid substitution comprising a R residue at position 94" would mean position 112 of the wild- type E. faecalis srtA as position 112 of the wild- type E. faecalis srtA corresponds to position 94 in the exemplary wild- type S . aureus srtA sequence (Gene ID: 3238307) when taking into account gaps and/or insertions in the alignment of E. faecalis srtA and S . aureus srtA.
  • the N residue at position 112 of the wild-type E. faecalis srtA amino acid sequence would be replaced by a R residue in the resulting mutant srtA.
  • any original wild-type sortase A sequence to be used for generating a calcium- independent srtA mutant with an exemplary wild-type S. aureus sortase A sequence for purposes of determining the positions in the original wild-type sortase A sequence that correspond to the exemplary wild-type S . aureus sortase A sequence when taking into account gaps and/or insertions in the alignment of the two sequences.
  • the substitution comprises a E105K substitution.
  • E105K substitution in reference to mutating a particular original sortase A sequence (e.g., a wild type sortase A sequence) refers to the substitution of a K residue at a position in the original sortase A amino acid sequence that corresponds to the E105 residue in the corresponding exemplary wild- type S. aureus srtA amino acid sequence .
  • the original sortase A sequence to be mutated in accordance with the disclosure is a L. monocytogenes srtA sequence (NCBI Gene ID:
  • the phrase "E105K substitution” refers to the substitution of the R residue at position 112 of the L. monocytogenes srtA sequence wi h a K residue as position 112 of L. monocytogenes srtA corresponds to position 105 of the exem lary wild-type S. aureus srtA sequence (NCBI Gene ID : 3238307) when the two sequences are aligned taking into account any gaps and/or insertions.
  • the substitution comprises a E108Q
  • substitution comprises an E108A substitution. In some embodiments, the substitution comprises a P94R substitution. In some embodiments, the substitution comprises a P94S
  • substitution comprises a D160N substitution. In some embodiments, the substitution comprises a D165A substitution. In some embodiments, the substitution comprises a K190E
  • the substitution comprises a K196T substitution.
  • the wild-type sortase A comprises S. aureus sortase A. In some embodiments, the wild-type sortase A comprises an amino acid sequence of
  • the wild-type sortase A comprises an amino acid sequence of SEQ ID NO: 3.
  • the calcium- independent srtA mutant comprises a deletion of amino acids 2-25. In some embodiments, the calcium- independent srtA mutant
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions selected from the group consisting of i) -vi) .
  • the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising i) and iii) . In some embodiments, the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising i) and iv) . In some embodiments , the calcium-independent srtA mutant
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising i) and vi) .
  • the calcium-independent srtA mutant comprises at least two amino acid substitutions comprising ii) and iii) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising ii) and iv) . In some embodiments, the calcium- independent srtA mutant
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising ii) and vi) .
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iii) and iv) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iii) and v) . In some embodiments, the calcium- independent srtA mutant
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least two amino acid substi utions comprising iv) and vi) .
  • the calcium- independent srtA mutant comprises at least two amino acid substitutions comprising v) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions selected from the group consisting of i) -vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and iv) . In some embodiments, the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iii) and vi) .
  • the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising i) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , iv) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising i) , v) and vi) .
  • the calcium-independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and iv) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iii) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , iv) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising ii) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iii) , iv) and v) . In some embodiments , the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iii), v) and vi) .
  • the calcium- independent srtA mutant comprises at least three amino acid substitutions comprising iv) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least four amino acid substitutions selected from the group consisting of i) -vi) .
  • the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising i) , iii), iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising i) , iii) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising ii) , iii) , iv) and v) . In some embodiments, the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising ii) , iii) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least four amino acid substitutions comprising iii) , iv) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least five amino acid substitutions selected from the group consisting of i) -vi) .
  • the calcium- independent srtA mutant comprises at least five amino acid substitutions comprising i) , iii) , iv) , v) and vi) .
  • the calcium- independent srtA mutant comprises at least five amino acid substitutions comprising ii), iii), iv), v) and vi) .
  • the calcium- independent srtA mutants comprises at least 60% identity to SEQ ID NO: 1, or at least 65% identity to SEQ ID NO: 1, or at least 70% identity to SEQ ID NO: 1, or at least 75% identity to SEQ ID NO: 1, or at least 80% identity to SEQ ID NO : 1, or at least 85% identity to SEQ ID NO: 1, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 1.
  • the calcium- independent srtA mutants comprises at least 60% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 65% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 70% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 75% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 80% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 85% identity to amino acid residues 60-206 of SEQ ID NO: 1, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to amino acid residues 60-206 SEQ ID NO: 1.
  • the calcium- independent srtA mutants comprises at least 60% identity to SEQ ID NO: 3, or at least 65% identity to SEQ ID NO: 3, or at least 70% identity to SEQ ID NO: 3, or at least 75% identity to SEQ ID NO: 3, or at least 80% identity to SEQ ID NO : 3, or at least 85% identity to SEQ ID NO: 3, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 3.
  • the calcium- independent srtA mutants comprises at least 60% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 65% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 70% identity to amino acid residues 60-206 of SEQ ID NO : 3, or at least 75% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 80% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 85% identity to amino acid residues 60-206 of SEQ ID NO: 3, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to amino acid residues 60-206 SEQ ID NO: 3.
  • the calcium-independent srtA mutants comprises at least 60% identity to SEQ ID NO : 5, or at least 65% identity to SEQ ID NO: 5, or at least 70% identity to SEQ ID NO: 5, or at least 75% identity to SEQ ID NO: 5, or at least 80% identity to SEQ ID NO: 5, or at least 85% identity to SEQ ID NO: 5, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or up to 99% identity to SEQ ID NO: 5.
  • any of the calcium- independent srtA mutants disclosed herein can include a tag.
  • a calcium-independent srtA mutant comprises a C-terminal tag.
  • a calcium- independent srtA mutant comprises a N-terminal tag.
  • the calcium- independent srtA mutant comprises a N-terminal His6 tag.
  • the calcium- independent srtA mutants exhibit sortase A catalytic activity in the absence of calcium. In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the absence of exogenous calcium.
  • the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium-binding proteins.
  • the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations up to 1 mM, up to 2 mM, up to 3 mM, up to 4 mM, up to 5 mM, up to 6 mM, up to 7 mM, up to 8 mM, up to 9 mM, or up to 10 mM or more.
  • the calcium-independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations less than 1 mM. In some embodiments, the calcium- independent srtA mutants exhibit sortase A catalytic activity in the presence of calcium concentrations less than 100 ⁇ , less than 10 ⁇ , less than 1 ⁇ , less than 0.1 ⁇ , or less than 0.01 ⁇ .
  • the calcium-independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of wild-type sortase A catalytic activity at 0 mM Ca 2+ compared to wild-type sortase A catalytic activity at 10 mM Ca 2+ .
  • the calcium-independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of wild-type sortase A catalytic activity at 0 mM Ca 2 ' ' compared to wild- type sortase A catalytic activity at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 1- fold, at least 2 - fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100-fold, or more sortase A catalytic activity at 0 mM Ca 2+ compared to wild-type sortase A catalytic activity at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca 2 " .
  • the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 1.
  • the calcium-independent srtA mutants exhibit at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100- fold, or more than sortase A catalytic activity of SEQ ID NO: 1 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 1 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 3.
  • the calcium- independent srtA mutants exhibit at least at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold, at least 20-fold, at least 30-fold, at least 40- fold, at least 50-fold, at least 60-fold, at least 70- fold, at least 80-fold, at least 90-fold, or at least up to 100-fold, or more than sortase A catalytic activity of SEQ ID NO: 3 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 3 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least up to 80% of sortase A catalytic activity of SEQ ID NO: 5 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 5 at 10 mM Ca 2+ .
  • the calcium- independent srtA mutants exhibit at least 85%, at least 90%, at least 91%, at least 92%, at least 90%, at least 91%, or at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to
  • the calcium- independent srtA mutants disclosed herein exhibit increased catalytic activity compared to the sortase A of SEQ ID NO: 5.
  • the calcium- independent srtA mutants exhibit at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least up to 100- fold, or more than sortase A catalytic activity of SEQ ID NO: 5 at 0 mM Ca 2+ compared to sortase A catalytic activity of SEQ ID NO: 5 at 10 mM Ca 2+ .
  • aspects of the present disclosure also relate to calcium- independent mutants comprising an amino acid sequence at least 80% identical to SEQ ID NO: 9.
  • a calcium- independent srtA mutant comprises an amino acid sequence at least 80% identical to SEQ ID NO : 9, wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO: 9; b) a Q residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO:9; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO : 9; and v) a T residue at position 138 of SEQ ID NO : 9.
  • a calcium- independent srtA mutant comprises an amino acid sequence at least 80% identical to SEQ ID NO : 9, wherein the mutant comprises a) a K residue at position 47 of SEQ ID NO : 9; b) a A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36 of SEQ ID NO: 9; ii) a N residue at position 102 of SEQ ID NO: 9; iii) a A residue at position 107 of SEQ ID NO: 9; iv) a E residue at position 132 of SEQ ID NO: 9; and v) a T residue at position 138 of SEQ ID NO: 9.
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 16 (FIG. 4P) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 16 comprising a A residue at position 50. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 17 (FIG. 4Q) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 17 comprising a A residue at position 50. In some
  • a calcium-independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 18 (FIG. 4R) .
  • a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 18 comprising a A residue at position 50.
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 19 (FIG. 4S) .
  • a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 19 comprising a A residue at position 50.
  • a calcium-independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 20 (FIG. 4T) .
  • a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 20 comprising a A residue at position 50.
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 21 (FIG. 4U) .
  • a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 21 comprising a A residue at position 50.
  • a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .
  • a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and ii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and iv) . In some embodiments, a calcium-independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising i) and v) .
  • a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iv) . In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO : 9 comprising ii) and v) .
  • a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO : 9 comprising iii) and iv) . In some embodiments , a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising iii) and v) .
  • a calcium- independent srtA mutant comprises at least two amino acid residues of SEQ ID NO: 9 comprising iv) and v) . In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 15 (FIG. 40) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 15 comprising a A residue at position 50.
  • a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .
  • a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and iii) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and iv) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) , and v) .
  • a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO: 9 comprising ii) , iii) , and iv) . In some embodiments, a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO : 9 comprising ii) , iv) , and v) .
  • a calcium- independent srtA mutant comprises at least three amino acid residues of SEQ ID NO : 9 comprising iii ) , iv) , and v) .
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 12. (FIG . 4L) ) In some embodiments , a calcium- independent srtA mutant comprises an amino acid sequence SEQ ID NO:
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO : 14 (FIG . 4N) .
  • a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 14 comprising a A residue at position 50.
  • a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .
  • a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising i) , ii) , iii) , and iv) . In some embodiments, a calcium-independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising i) , iii) , iv) , and v) . In some embodiments, a calcium- independent srtA mutant comprises at least four amino acid residues of SEQ ID NO: 9 comprising ii) , iii), iv) , and v) .
  • a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 11 (FIG. 4K) . In some embodiments, a calcium independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 11 comprising a A residue at position 50.
  • a calcium-independent srtA mutant comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, a calcium-independent srtA mutant comprises an amino acid sequence at least 96% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 97% identical to SEQ ID NO: 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 98% identical to SEQ ID NO : 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence at least 99% identical to SEQ ID NO : 9. In some embodiments, a calcium- independent srtA mutant comprises an amino acid sequence of SEQ ID NO: 9.
  • polynucleotides encoding mutants of sortase A comprising a nucleotide sequence at least 80% identical to SEQ ID NO: 10 are disclosed.
  • a nucleotide sequence at least 80% identical to SEQ ID NO: 10 are disclosed.
  • polynucleotide encoding a mutant of sortase A comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 10, and encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a Q residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group
  • a polynucleotide encoding a mutant of sortase A comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 10, and encodes a) a K residue at position 47 of SEQ ID NO: 9; b) a A residue at position 50 of SEQ ID NO: 9; and c) at least one amino acid residue of SEQ ID NO: 9 selected from the group consisting of i) a R residue at position 36; ii) a N residue at position 102; iii) a A residue at position 107; iv) a E residue at position 132; and v) a T residue at position 138.
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and ii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and iii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and iv) . In some
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising i) and v) .
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iii) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and iv) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising ii) and v) .
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iii) and iv) . In some embodiments, the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iii) and v) .
  • the polynucleotide encodes at least two amino acid residues of SEQ ID NO: 9 comprising iv) and v) .
  • the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9
  • the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) and iv) . In some embodiments, the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising i) , ii) and v) .
  • the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9
  • the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9 comprising ii) , iii) and v) .
  • the polynucleotide encodes at least three amino acid residues of SEQ ID NO: 9
  • the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 selected from the group consisting of i) -v) .
  • the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 comprising i) , ii) , iii) , and iv) . In some embodiments , the polynucleotide encodes at least four amino acid residues of SEQ ID NO: 9 comprising ii) , iii) , iv) and v) .
  • the polynucleotide encodes at each of the amino acid residues of SEQ ID NO: 9.
  • a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 85% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 10.
  • a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 10. In some embodiments, polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 10.
  • a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence at least 100% identical to SEQ ID NO: 10. In some embodiments, a polynucleotide encoding a calcium- independent srtA mutant comprises a nucleotide sequence of SEQ ID NO: 10.
  • nucleic acid construct comprising a polynucleotide disclosed herein (e.g., a polynucleotide encoding a calcium- independent srtA mutant) .
  • the nucleic acid construct comprises a nucleotide sequence that encodes one or more C-terminal or N-terminal tags.
  • polynucleotide refers to a molecule, which is a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single stranded or double stranded.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • the polynucleotides of the present disclosure such as polynucleotides encoding the calcium- independent srtA mutants, can be isolated or synthesized using standard molecular biology techniques and the sequence information provided herein. The synthetic
  • polynucleotides may be optimized in codon use, preferably according to the methods described in WO2006/077258 and/or PCT/EP2007/055943 , which are herein incorporated by reference.
  • PCT/EP2007/055943 addresses codon-pair optimization.
  • the polynucleotides encoding the calcium- independent srtA mutants of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PGR amplification techniques. The nucleic acid so amplified can be cloned into an
  • a polynucleotide may either be present in isolated form, or be comprised in recombinant nucleic acid molecules or vectors, or be comprised in a host cell.
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native
  • polypeptides and proteins expressed in host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single- step purification method
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Expression vectors useful in the present disclosure include chromosomal-, episomal- and virus-derived vectors e.g., vectors derived from bacterial plasmids,
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • yeast episome yeast chromosomal elements
  • viruses such as baculoviruses , papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses
  • vectors derived from combinations thereof such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids .
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic
  • the vectors, such as expression vectors, of the disclosure can be introduced into host cells to thereby produce proteins or peptides, encoded by nucleic acids as described herein (e.g. calcium- independent srtA mutant proteins, catalytically active fragments, catalytically active variants or catalytically active derivatives thereof) .
  • the vectors, such as recombinant expression vectors, of the disclosure can be designed for expression of calcium-independent srtA mutant proteins in
  • prokaryotic or eukaryotic cells prokaryotic or eukaryotic cells.
  • calcium- independent srtA mutant proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression
  • a "host cell,” as used herein, is any cell capable of being grown and maintained in cell culture under conditions allowing for production and recovery of useful quantities of a biological product, as defined herein.
  • Host cells can be unmodified cells or cell lines, or cell lines which have been genetically modified (e.g., to facilitate production of a biological product) .
  • the host cell is a cell line that has been modified to allow for growth under desired conditions, such as in serum-free media, in cell suspension culture, or in adherent cell culture.
  • a host cell can be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in a specific, desired fashion . Such modifications (e.g. , glycosylation) and processing (e.g., cleavage) of protein products may facilitate optimal functioning of the protein.
  • Suitable host cells are preferably prokaryotic microorganisms such as bacteria (e.g., E. coli) , or in some embodiments eukaryotic organisms, for example f ngi, such as yeasts or filamentous fungi, or plant cells.
  • the calcium- independent srtA mutant according to the disclosure can be recovered and purified from recombinant cell cultures by methods known in the art (e.g., ion- exchange chromatography, hydrophobic interaction
  • exclusion chromatography to further separate the target calcium- independent mutant srtA from the bulk protein to enable recovery of the target calcium- independent srtA mutant in a highly purified state
  • the disclosure relates to a method of producing a calcium- independent srtA mutant comprising the steps of: (a) culturing the host cell according to the disclosure in a suitable culture medium under suitable conditions to produce calcium- independent srtA mutant; and optionally (b) purifying said calcium- independent srtA mutant to provide a purified calcium- independent srtA mutant product .
  • Another aspect of the present disclosure relates to methods for the production of substantially purified calcium- independent srtA mutant enzyme of the present disclosure. It should be appreciated that such enzyme can be prepared in compliance with Good Manufacturing Practices (GMP) or used in a GMP-com liant process.
  • GMP Good Manufacturing Practices
  • substantially purified calcium- independent srtA mutant involves cloning a nucleic acid segment encoding the calcium- independent srtA mutant enzyme can be inserted in a vector that contains sequences allowing expression of a sortase-transamidase in another organism, such as E.
  • a suitable host organism can then be transformed or transfected with the vector containing the cloned nucleic acid segment. Expression is then performed in that host organism. The expressed enzyme is then purified using standard techniques. Techniques for the purification of cloned proteins are well known in the art and need not be detailed further here (e.g., affinity chromatography on a nickel NTA column for purification of a calcium- independent srtA mutant enzyme extended at its carboxyl terminus with a sufficient number of histidine residues to allow specific binding of the protein molecule to the nickel NTA column through the histidine residues) .
  • the disclosure further provides an enzyme composition comprising one or more calcium- independent srtA mutants.
  • the disclosure relates to a sortase-mediated transpeptidation reaction catalyzed by an enzyme composition according to the disclosure. Examples of synthetic nucleophiles that can be used in such sortase-mediated transpeptidation reactions are shown in Table 1 below .
  • Table 1 Examples of synthetic nucleophiles used in site-specific sortase A transpeptidation reactions .
  • an enzyme composition disclosed herein for the sortagging of a protein.
  • An enzyme composition of the disclosure may comprise a polypeptide which has the same enzymatic activity, for example the same type of transamidase activity as that provided by a polypeptide of the disclosure.
  • An enzyme composition of the disclosure may comprise a polypeptide which has a different type of enzymatic activity than that provided by a polypeptide of the disclosure.
  • composition is purified to comprise calcium- independent srtA mutants of a particular sequence (e.g., SEQ ID NO: 9) .
  • enzyme composition is purified to comprise calcium- independent srtA mutants of a particular sequence (e.g., SEQ ID NO: 9) .
  • the enzyme composition is purified to comprise calcium- independent srtA mutants of a particular sequence (e.g., SEQ ID NO: 9) .
  • the enzyme composition is purified to comprise calcium- independent srtA mutants of a particular sequence (e.g., SEQ ID NO: 9) .
  • the enzyme composition comprises calcium- independent srtA mutants predominantly in monomeric form.
  • the enzyme composition comprises a mixture of both monomeric and dimeric calcium- independent srtA mutants .
  • the enzyme composition comprises a mixture of calcium- independent srtA mutants comprising different sequences.
  • the enzyme composition comprises at least two, at least three , at least four, or at least five calcium- independent sortase A mutants selected from the group consisting of SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 , SEQ ID NO: 13 , SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 , SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , and combinations thereof .
  • the enzyme composition comprises a group of calcium- independent sortase A mutants comprising SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:
  • the enzyme composition comprises a calcium- independent srtA mutant and a calcium-dependent srtA
  • the enzyme composition is a mixture of the enzyme composition
  • the enzyme composition comprises a
  • the enzyme composition is a mixture of the enzyme composition
  • the enzyme comprises: 15 NO: 3 or SEQ ID NO: 5. In some embodiments, the enzyme
  • composition comprises an aqueous medium.
  • the calcium- independent srtA mutants and enzyme compositions comprising the calcium- independent srtA
  • Exemplary such applications include, but are not limited to , specific incorporation of novel functionality into proteins , synthesis of neoglycoconjugates , immobilization of proteins to solid surfaces, protein labeling on living
  • eGFP-LPETG Polystyrene beads Parthasart hy et al . (2007) eGFP-LPETGG-His f , Glycidyl methacrylate (GMA) Chan et al. (2007)
  • the calcium- independent srtA mutants according to the disclosure may feature a number of significant
  • these advantages may include aspects such as lower production costs,
  • a calcium- independent srtA mutant or composition of the disclosure may be used in any process which requires the sortagging of a moiety of interest to a target protein .
  • sortagging , “sortase- mediated ligation”, “sortase-mediated transpeptidation” , “sortase-mediated transacylation” , are used
  • tags include, but are not limited to, amino acids, peptides, proteins, nucleic acids, polynucleotides, sugars, carbohydrates, polymers, lipids, fatty acids, and small molecules. Other suitable tags will be apparent to those of skill in the art and the disclosure is not limited in this aspect.
  • a tag comprises a sequence useful for purifying, expressing, solubilizing, and/or detecting a polypeptide.
  • a tag comprises an HA, TAP, Myc, 6XHis, Flag, or GST tag, to name few examples .
  • a tag comprises a solubility-enhancing tag (e.g., a SUMO tag, NUS A tag, SNUT tag, a Strep tag, or a monomeric mutant of the Ocr protein of bacteriophage T7) . See, e.g., Esposito D and Chatterjee DK. Curr Opin Biotechnol . ; 17(4):353-8 (2006).
  • a tag is cleavable, so that it can be removed, e.g., by a protease. In some embodiments, this is achieved by including a protease cleavage site in the tag, e.g., adjacent or linked to a functional portion of the tag.
  • exemplary proteases include, e.g., thrombin, TEV protease, Factor Xa, PreScission protease, etc.
  • a "self-cleaving" tag is used. See, e.g., PCT/US05/05763.
  • a tag comprises a click chemistry handle.
  • the calcium-independent srtA mutants disclosed herein can be used to sortag cells to be administered to a subject, e.g., human subjects.
  • a method for sortagging a target polypeptide comprises (a) providing a polypeptide comprising a sortase recognition motif; (b) providing a moiety comprising an N-terminal oligoglycine sequence or a terminal alkylamine; and (c) contacting the target polypeptide with the moiety in the presence of an enzyme composition described herein (e.g., a composition comprising at least one calcium- independent srtA mutant disclosed herein) under conditions suitable for the srtA A mutant to ligate the moiety to the target protein, thereby sortagging the target protein.
  • an enzyme composition described herein e.g., a composition comprising at least one calcium- independent srtA mutant disclosed herein
  • sortagging methods disclosed herein can be used to attach a moiety to any target protein or polypeptide.
  • Methods and compositions provided herein can be used to conjugate essentially any polypeptide to any moiety.
  • Non- limiting examples of polypeptides that can be produced or conjugated according to methods provided herein include receptors, membrane proteins, cytokines, chemokines, hormones, enzymes, growth factors, growth factor receptors, antibodies, antibody derivatives and other immune effectors,
  • interleukins interleukins , interferons, erythropoietin, integrins, soluble major histocompatibility complex antigens, binding proteins, transcription factors, translation factors, oncoproteins or proto-oncoproteins , muscle proteins, myeloproteins, neuroactive proteins, tumor growth suppressors, structural proteins, and blood proteins (e.g., thrombin, serum albumin, Factor VII, Factor VIII, Factor IX, Factor X, Protein C, von
  • the polypeptide is a glycoprotein or other polypeptide which requires post- translational modification, such as deamidation, glycation, or the like, for optimal activity.
  • the polypeptide is a lipoprotein. Exemplary target polypeptides which have been sortagged are shown in Table 3 below.
  • Table 3 Examples of proteins labeled by sortase A transpeptidation .
  • the target polypeptide comprises or consists of a polypeptide that is at least 80%, or at least 90%, e.g., at least 95%, 86%, 97%, 98%, 99%, 99.5%,
  • the target polypeptide has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences relative to a naturally occurring sequence.
  • the naturally occurring protein is a mammalian protein, e.g., of human origin.
  • Naturally occurring sequences e.g., genomic, mRNA, and polypeptide sequences, from a wide variety of species, including human, are known in the art and are available in publicly accessible databases such as those available at the National Center for Biotechnology Information (www.ncbi.nih.gov) or Universal Protein Resource
  • Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt , UniProtKB/Trembl , and the like. Sequences, e.g., nucleic acid (e.g., mRNA) and polypeptide sequences, in the NCBI Reference Sequence database may be used as reference sequences.
  • a target polypeptide is a protein that is approved by the US Food & Drug
  • Such proteins may or may not be one for which a PEGylated version has been tested in clinical trials and/or has been approved for marketing .
  • a target polypeptide is a neurotrophic factor, i.e., a factor that promotes survival , development and/or function of neural lineage cells (which term as used herein includes neural
  • progenitor cells e.g., astrocytes, oligodendrocytes, microglia.
  • glial cells e.g., astrocytes, oligodendrocytes, microglia
  • t e target protein is one that forms homodimers or heterodimers , (or homo- or heterooligomers comprising more than two subunits, such as tetramers) .
  • the target polypeptide is an enzyme, e.g., an enzyme that is important in metabolism or other physiological processes.
  • a target protein comprises a receptor or receptor fragment (e.g., extracellular domain).
  • the target polypeptide is sortagged in cells to be administered to a subject.
  • the present disclosure contemplates any application for which administration of cells comprising a sortagged
  • polypeptide is desirable (e.g., therapeutic, diagnostic, imaging, etc.)
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal.
  • Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice , rats, woodchucks , ferrets , rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species , e.g., chicken, emu, ostrich, and fish, e.g., trout , catfish and salmon .
  • Subj ect includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans , primates or rodents .
  • the subj ect is a mammal, e.g., a primate, e.g., a human .
  • the subj ect is a mammal .
  • the mammal can be a human, non- human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used, for example, as subj ects that represent animal models of disease .
  • a subj ect can be male or female .
  • proteins e.g., secreted eukaryotic (e.g., mammalian) proteins
  • intracellular processing e.g., cleavage of a secretion signal prior to secretion and/or removal of other portion (s) that are not required for biological activity
  • Such mature, biologically active versions of target proteins are used in certain embodiments of the disclosure.
  • polypeptide according to methods provided herein can be any agent suitable for conjugation to a polypeptide, i.e., capable of being operably linked to a sortase recognition sequence.
  • the moiety can confer any of a number of possible functionalities to the polypeptide, such as but not limited to, altered physico-chemical properties, such as solubility and/or stability,- altered pharmacokinetic properties, such as bioavailability, clearance rate, and/or plasma half-life and/or altered biological activity, such as immunogenicity and/or antigenicity.
  • Non-limiting examples of moieties include: a small -molecule , a peptide, a polypeptide, a lipid and/or fatty acid, a carbohydrate, a nucleic acid, a reporter molecule (e.g., a reporter enzyme, fluorescent molecule, a radiolabel, an affinity label, or the like), a toxin, a therapeutic agent, a nanoparticle, a resin, a cell, a virus particle, an adjuvant molecule, or a polymer (e.g., a hydrophilic polymer), an affinity tag (e.g., His6) , or the like.
  • the moiety is a pharmacological carrier molecule.
  • the moiety comprises, consists essentially of, or consists of a member of a prosthetic binding group, such as biotin/avidin,
  • biotin/streptavidin maltose binding protein/maltose
  • glutathione S-transferase/glutathione glutathione S-transferase/glutathione
  • metal/polyhistidine antibody/epitope , antibody/antigen, antibody/protein A or protein G, hapten/anti-hapten, folic acid/folate binding protein, vitamin B 12/intrinsic factor, nucleic acid/complementary nucleic acid,
  • the moiety comprises, consists essentially of, or consists of a peptide, a
  • peptidomimetic e.g., a peptoid
  • an amino acid an amino acid analog
  • a polynucleotide or polynucleotide analog e.g., a polynucleotide or a nucleotide or nucleotide analog
  • an organic or inorganic compound having a molecular weight between about 500 and about 10,000.
  • the moiety comprises, consists essentially of, or consists of a second polypeptide.
  • the polypeptide can be any polypeptide.
  • a protein which is difficult to produce in a cell e.g., either due to toxicity
  • fragments which can be j oined using the methods described herein e.g. , first portion of the protein can be attached to the second portion, reconstituting an active protein using the methods described herein.
  • the moiety comprises , consists essentially of , or consists of a reporter molecule , such as a fluorescent molecule (e.g. , umbelliferone ,
  • fluorescein fluorescein isothiocyanate , rhodamine , dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • a radioisotope e.g., Cu-64, Ga67, Ga-68, Zr-89, Ru-97, Tc-99, Rh-105, Pd-109, In- 1.1.1, 1-123 , I- 125, 1-131, Re-186, Re- 188, Au-198 , Pb-203 , At-211, Pb- 212 or Bi-212
  • a detectable enzyme e.g.
  • horseradish peroxidase alkaline phosphatase, p-galactosidase, or acetylcholinesterase
  • a luminescent material e.g., luminol
  • a bioluminescent material e.g., luciferase, luciferin, or aequorin
  • the moiety comprises, consists essentially of, or consists of a biologically active molecule, such as a toxin (e.g., abrin, ricin A,
  • pseudomonas exotoxin or diphtheria toxin .
  • the moiety comprises the polypeptide itself, such that the polypeptide is cyclized by the conjugation.
  • cyclized proteins often exhibit desired properties relative to the
  • polypeptide can be 'chained' (e.g., dimerized, trimerized, etc) .
  • the moiety is a water-soluble polymer, non-peptidic polymer with an average molecular weight of about 200 to about 200,000 Daltons, depending on the desired effect on the properties of the
  • the moiety comprises, consists essentially of, or consists of a polymeric group, such as polyalkylene oxide (PAO) , polyalkylene glycol (PAG) , polyethylene glycol (PEG) , methoxypolyethylene glycol (mPEG) , polypropylene glycol (PPG), branched PEGs, copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol (PVA) ,
  • PAO polyalkylene oxide
  • PAG polyalkylene glycol
  • PEG polyethylene glycol
  • mPEG methoxypolyethylene glycol
  • PPG polypropylene glycol
  • PVA polyvinyl alcohol
  • polycarboxylate poly-vinylpyrrolidone , polyethylene-co- maleic acid anhydride, polystyrene-co-maleic acid anhydride, dext rboxymethy1 -dextran,
  • polyoxyethylated glycerol polyoxyethylated sorbitol, polyoxyethylated glucose, dextran, polyoxazoline, polyacryloylmorp oline, or a serum protein binding- ligand, such as a compound which binds to albumin (e.g., fatty acids, C 5 -C 2 4 fatty acid, aliphatic diacid (e.g. C 5 - C 24 ) ) .
  • albumin e.g., fatty acids, C 5 -C 2 4 fatty acid, aliphatic diacid (e.g. C 5 - C 24 )
  • compositions provided herein are known in the art and are described, e.g., in U.S. Pat. No. 5,629,384, which is herein incorporated by reference.
  • the target polypeptide or moiety comprises an affinity tag that can be used to facilitate recovery and/or isolation of the conjugated polypeptide.
  • An affinity tag used in a method or composition provided herein can comprise any peptide or other molecule for which an antibody or other specific binding agent is available.
  • Affinity tags known in the art as being useful for protein purification include, but are not limited to, a poly-histidine segment, protein A (e.g., Nilsson et al . , EMBO J. 4:1075 (1985); Nilsson et al . , Methods Enzymol . 198:3 (1991)), glutathione S transferase (e.g., Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (e.g., Grussenmeyer et al . , Proc . Natl. Acad. Sci. USA 82:7952 (1985)), substance P, FLAG peptide (e.g., Hopp et al . , Biotechnology 6:1204 (1988)), c-myc tags (detected with anti-myc antibodies) ,
  • an affinity tag described herein allows for selective enrichment of desired conjugation products.
  • an affinity tag is located N- terminal of a sortase recognition sequence or C- terminal of an oligoglycine sequence so that the tag remains associated with the polypeptide after sortase- catalyzed cleavage and ligation. As such, the affinity tag is retained in the conjugated polypeptide upon cleavage and/or ligation of the sortase recognition sequence by a sortase and affinity
  • an affinity tag is located C- terminal of a sortase recognition sequence so that the tag is cleaved from the polypeptide upon sortase- catalyzed cleavage and ligation.
  • the affinity tag is located C-terminal of the sortase recognition sequence (i.e., the sortase recognition sequence is between the target polypeptide and the affinity tag) .
  • the target polypeptide comprises a spacer peptide.
  • a spacer peptide separates the target polypeptide from a sortase recognition sequence and/or an affinity tag, and/or the sortase recognition sequence from an affinity tag.
  • a spacer peptide can be of any size, e.g., from several to 30 or more amino acid residues, sufficient to serve the intended purpose. Spacer peptides can enhance conformational flexibility between two or more domains of a protein and/or minimize steric interference with the folding and/or function of two or more domains of a protein.
  • a spacer peptide will generally comprise an inert, flexible amino acid sequence, e.g., comprising predominantly glycine, serine, and/or alanine residues.
  • a spacer peptide sequence can be modified with one or more proline residues at the beginning and/or at the end of the spacer in order to isolate the spacer as a separate functional domain from neighboring domains of the protein.
  • spacer peptides are known in the art.
  • Contacting refers to the addition of the target polypeptide to the culture medium in a manner that allows the calcium- independent srtA mutant to ligate the moiety to target polypeptide.
  • the contacting includes culturing the cells for a defined period of time in the presence of the target polypeptide. In other embodiments, the contacting includes culturing the cells for a variable period of time until a desired endpoint or other indicator is achieved.
  • Conditions which allow the calcium- independent srtA mutant to cleave the sortase recognition sequence and ligate the moiety to the target polypeptide include for example, standard cell growth conditions known to those of skill in the art, e.g. for mammalian cells; 37° C . , 5% C0 2 , and an appropriate cell culture medium.
  • the cell culture medium may vary depending upon the host cell and can be determined readily by those of skill in the art.
  • the "contacting" step in the method takes place in the absence of calcium, i.e., there is no exogenous calcium added to the culture medium or reaction mixture .
  • a sorta.se is immobilized by attaching it to a support.
  • a "support” may be any entity or plurality of entities having a surface to which a substance may be attached or on which a substance may be placed. Examples, include, e.g., particles, slides, filters, interior wall or bottom of a vessel (e.g., a culture vessel such as a plate or flask, well of a microwell plate, tube), chips, etc.
  • a support may be composed, e.g., of glass, metal, gels (e.g., agarose) , ceramics, polymers, or combinations thereof.
  • Immobilization may comprise contacting sortase or a composition containing sortase with an affinity reagent, e.g., an antibody, that binds to sortase, wherein the affinity reagent is attached to a support.
  • an affinity reagent e.g., an antibody
  • the sortase is tagged, and the affinity reagent binds to the tag.
  • sortase may comprise a tag, e.g., a 6X-His tag, which may be used to immobilize the sortase to a metal-ion containing resin or substrate.
  • sortase is immobilized to magnetic particles.
  • magnetic particles may be magneti sable and paramagnetic, e.g., superparamagnetic, i.e., they may only magnetic in a magnetic field.
  • the support is in a column. Unreacted sortase substrates and reaction products may readily be separated from an immobilized sortase.
  • the disclosure encompasses agents produced according to methods described herein, and compositions comprising such agents. It will be understood that, in some aspects, the disclosure encompasses methods of using such agents, e.g., for one or more purposes described herein.
  • the disclosure further provides packaged products and kits, including calcium- independent srtA mutants described herein or polynucleotides encoding the same, cell lines, cell cultures, populations and compositions, including, as well cells, cultures, populations, and compositions enriched or selected for any calcium- independent srtA mutants or variants thereof, packaged into suitable packaging material.
  • a packaged product or kit includes calcium- independent srtA mutants in monomeric form.
  • the packaged product or kit includes calcium-independent srtA mutants in dimeric form.
  • the packaged product or kit includes a mixed population of monomeric and dimeric calcium- independent srtA mutants.
  • a packaged product or kit includes a label, such as a list of the contents of the package, or instructions for using the kit e.g., instructions for sortagging a target polypeptide, isolating or producing a substantially purified calcium- independent srtA mutant disclosed herein, administering sortagged cells, e.g., implanting or transplanting in vivo, or screening for a compound or agent that modulates activity of the calcium- independent srtA mutants or variants thereof .
  • a label such as a list of the contents of the package
  • instructions for using the kit e.g., instructions for sortagging a target polypeptide, isolating or producing a substantially purified calcium- independent srtA mutant disclosed herein, administering sortagged cells, e.g., implanting or transplanting in vivo, or screening for a compound or agent that modulates activity of the calcium- independent srtA mutants or variants thereof .
  • a packaged product or kit includes a container, such as a sealed pouch or shipping container, or an article of manufacture, for example, to carry out a sortase-mediated ligation reaction utilizing a calcium-independent srtA mutant described herein, variant thereof or composition comprising the same, or preserving or storing the calcium- independent srtA mutants , such as a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8 , 16, 32 , 64, 96, 384 and 1536 multi- well plate or dish) .
  • a container such as a sealed pouch or shipping container, or an article of manufacture, for example, to carry out a sortase-mediated ligation reaction utilizing a calcium-independent srtA mutant described herein, variant thereof or composition comprising the same, or preserving or storing the calcium- independent srtA mutants , such as a tissue culture dish, tube, flask, roller bottle
  • packaging material refers to a physical structure housing the product or components of the kit .
  • the packaging material can maintain the components sterilel , and can be made of material commonly used for such purposes (e.g. , paper, corrugated fiber, glass , plastic , foil , ampules , etc . ) .
  • a label or packaging insert can be included, listing contents or appropriate written instructions, for example, practicing a method of the disclosure.
  • a packaged product or kit can therefore include instructions for practicing any of the methods of the disclosure described herein.
  • calcium- independent srtA mutants described herein, variants thereof, or enzyme compositions comprising them can be included in a tissue culture dish, tube, flask, roller bottle or plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) together with instructions, e.g., for sortagging, purification, preserving or screening.
  • Instructions may be on "printed matter," e.g., on paper or cardboard within the kit, on a label affixed to the package, kit or packaging material, or attached to a tissue culture dish, tube, flask, roller bottle, plate (e.g., a single multi-well plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well plate or dish) or vial containing a component of the kit.
  • Instructions may comprise voice or video tape and additionally be included on a computer readable medium, such as a disk (floppy diskette or hard disk) , optical CD such as CD- or DVD- ROM/RAM, magnetic tape, electrical storage media such as RAM and ROM and hybrids of these such as magnetic/optical storage media.
  • kits can optionally include additional components, such as buffering agent, a preservative, or a reagent.
  • additional components such as buffering agent, a preservative, or a reagent.
  • Each component of the kit can be enclosed within an individual container or in a mixture and all of the various containers can be within single or multiple packages .
  • nucleophiles can be used in a sortase reaction that comprise reactive chemical moieties, for example, moieties, or "handles", suitable for a click chemistry- reaction, as is described in detail in Published PCT International Application WO 2013/036630, the entirety of which is incorporated herein by reference.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • any one or more nucleic acids , polypeptides , cells , species or types of organism, disorders, subjects, or combinations thereof, can be excluded .
  • composition of matter e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal
  • composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
  • claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
  • the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise.
  • invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum.
  • Numerical values include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by "about” or “approximately” , the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by "about” or “approximately” , the invention includes an embodiment in which the value is prefaced by "about” or “approximately” .
  • Approximately or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value) . It should be
  • Example 1 A substantially pure, stable, high yield, calcium-independent srtA monomeric mutant
  • the present inventors surprisingly and unexpectedly found that a calcium- independent srtA mutant (SEQ ID NO: 9) purifies predominantly in monomeric form compared to a calcium-dependent srtA mutant (SEQ ID NO: 7) that purifies both in dimeric and monomeric forms.
  • the sortase purification process involves direct loading of clarified E.coli lysate onto a nickel NTA column. His-tagged sortase binds to the nickel NTA column (via metal chelation) and is eluted using an imidazole buffer. Eluted material from the nickel NTA column is loaded onto a size exclusion chromatography column (SEC) (e.g., a HILOAD ® 16/600 SUPERDEX ® 75 pg) and resulting separations (chromatogram) are show in FIGS. 7A, 7B and 7C.
  • SEC size exclusion chromatography column
  • the column resolves/separates residual high
  • the calcium- independent srtA mutant of SEQ ID NO: 9 exists predominantly in monomeric form (FIG. 7A) , in contrast, as noted above, the calcium-dependent sortase obtained from SEQ ID NO: 7 exists in both dimeric and monomeric form (FIG. 7B) , as does the calcium- dependent wild-type sortase (FIG. 7C) .
  • Those skilled in the art will appreciate the unexpected advantages which this confers on the calcium- independent srtA mutant of SEQ ID NO: 9, including a higher recovery yield of 100% monomer, and simpler manufacturing process to isolate monomer as there is far less dimer to separate out, amongst others.
  • the lower amount of dimer form observed with the calcium- independent srtA mutant of SEQ ID NO: 9 is due to lower propensity to dimerize based on the outer surface location of the substituted amino acids between the calcium-dependent srtA mutant of SEQ ID NO: 7 and the calcium- independent srtA mutant of SEQ ID NO: 9 (see, e.g., FIG. 5B for an alignment of SEQ ID NO: 7 and SEQ ID NO: 9).
  • the lower propensity of the calcium-independent srtA mutant of SEQ ID NO: 9 to dimerize is likely to translate into a more "stable" enzyme as it should also have a lower tendency to dimerize during storage, which may prolong shelf-life or permit storage under less costly conditions (e.g. refrigerated vs. frozen at - 80°C) .
  • FIG. 8 shows a gel demonstrating the high purity of monomeric sortase compared to dimeric sortase.
  • the gel illustrates the protein constituents present in the three main components from the calcium- independent srtA mutant of SEQ ID NO: 9 preparation separated on the SEC purification column. Collected fractions were pooled comprising the Void, Dimer and Monomeric peaks shown in FIG. 7B.
  • the "Load” in FIG. 8 represents the starting material in the clarified E. coli lysate.
  • the "Void" lane in FIG. 7B contains some sortase along with a large number of higher molecular weight proteins.
  • the "Dimer” lane in FIG. 8 contains
  • FIG. 9 shows the SEC results demonstrating the stability of monomeric sortase. Briefly, the dimeric and monomeric pools collected from the SEC purification step described above were stored at 4 °C for 24 hours, and then analyzed by analytical SEC (SUPERDEX ® 75 10/300 GL) . As shown in FIG.
  • the calcium- independent srtA mutant of SEQ ID NO: 9 was generated by introducing two additional nucleotide point mutations into a calcium-dependent srtA mutant of SEQ ID NO: 7. These nucleotide point mutations comprised a G->A mutation at position 139, and a G-C mutation at position 148 of SEQ ID NO: 7 .
  • the mutations were introduced on primers used to amplify a pET29 mutant delta 59 SrtA construct (see, e.g., Liu et al . , "A general strategy for the evolution of bond-forming enzymes using yeast display," PNAS.
  • PCR amplification was then be performed in two stages. First, the construct was amplified with primers shown in Table 4 below such that the forward primer introduced a restriction site for cloning (e.g., a Ndel restriction site), and the reverse primer introduced the two point mutations described above .
  • a restriction site for cloning e.g., a Ndel restriction site
  • the reverse primer introduced the two point mutations described above .
  • the second half of the construct was amplified with the primers shown in Table 5 below such that the forward primer introduced the two point mutations described above, and the reverse primer introduced a restriction site for cloning (e.g., a Xhol restriction site) .
  • a restriction site for cloning e.g., a Xhol restriction site
  • PCR reactions were performed using the pET29 pentamutant delta 59 SrtA plasmid DNA as a template, under the conditions set forth in Table 6 and cycling conditions set forth in Table 7 below.
  • the resulting PGR products from the first round of PGR were analyzed by gel electrophoresis, excised from the gel and purified. The products were then used as a template in a second round of PGR, and amplified using forward primer 1 and reverse primer 2 described above.
  • the second round of PCR reactions was carried out under the conditions set forth in Table 8 below under the cycling conditions set forth in Table 9.
  • the resulting product from the second round of PGR reactions was analyzed by gel electrophoresis, digested with Ndel and Xhol , and ligated into pET30b so that there is 6x his tag at the 3' end of the construct.
  • SEQ ID NO: 1 Exemplary Wild Type S. aureus Sortase A amino acid sequence
  • SEQ ID NO: 3 Exemplary Wild Type S. aureus Sortase A amino acid sequence

Abstract

Cette invention concerne des mutants de sortase A calcium-indépendants manifestant une activité catalytique accrue comparativement à la sortase A de type sauvage.
PCT/US2015/017116 2014-02-21 2015-02-23 Mutants de sortase a calcium-indépendants WO2015127365A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018005716A3 (fr) * 2016-07-01 2018-02-08 Denali Therapeutics Inc. Variants de l'albumine pour une demi-vie sérique améliorée
US20220403360A1 (en) * 2021-01-28 2022-12-22 Genequantum Healthcare (Suzhou) Co., Ltd. Ligase fusion proteins and application thereof

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AU784043B2 (en) * 1999-04-15 2006-01-19 Regents Of The University Of California, The Identification of sortase gene
US9588110B2 (en) * 2011-07-28 2017-03-07 Cell Signaling Technology, Inc. Multi component antibody based detection technology

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018005716A3 (fr) * 2016-07-01 2018-02-08 Denali Therapeutics Inc. Variants de l'albumine pour une demi-vie sérique améliorée
US20220403360A1 (en) * 2021-01-28 2022-12-22 Genequantum Healthcare (Suzhou) Co., Ltd. Ligase fusion proteins and application thereof

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